Detection of Nucleic Acids
There are many situations in which it is necessary to detect, either qualitatively or quantitatively, the presence of nucleic acids such as DNA and RNA or their constituent nucleotides. Examples of such situations include medical diagnosis (eg, the detection of infectious agents like bacteria and viruses, the diagnosis of inherited and acquired genetic diseases and the establishment of tissue type), forensic tests in criminal investigations and paternity disputes and of course the more general attempt to sequence human and animal genes.
Techniques are already known for detecting nucleic acids and nucleic acid units. Available methods include, for instance:
a) fluorescence spectroscopy--this is technically very demanding if high sensitivities are to be achieved. In biological assays, its use tends to be complicated by autofluorescence of the analytes.
b) radiolabelling--this also requires high levels of technical skill but tends to be less sensitive than fluorescence spectroscopy. It also suffers from the obvious hazards involved in handling radioactive materials.
c) chemiluminescence--although this technique can be relatively quick to carry out, and avoids the problem of autofluorescence and the need to handle toxic substances, it is unfortunately relatively insensitive and yet is still technically demanding.
A disadvantage common to many known techniques is their need for large amounts of the target analyte, ie, their relatively low sensitivity. Often in the situations mentioned above the target is simply not available in sufficiently high concentrations. As a result, the available target material has to be amplified before its presence can be accurately detected.
Again, techniques are known for amplifying a nucleic acid. The most common is the well-known "polymerase chain reaction" ("PCR"). Alternatively, the target nucleic acid may be cloned into a biological vector such as a plasmid, a phage or the like, which is then inserted into a (typically bacterial) host cell. The host is permitted to multiply and the desired vector is "harvested" from the host cell after an appropriate period of time.
Clearly, the need for amplification makes a detection method more complex, costly and time-consuming and introduces greater potential for error and for contamination of the target material.
There is therefore a need for a nucleic acid detection method which is sensitive to relatively low target concentrations, and which can preferably be carried out directly on an unamplified sample. It is this need that the present invention addresses.
Surface Enhanced Raman Scattering
The invention provides a technique based on the principle of "surface enhanced Raman scattering" (SERS) and on a modification of that principle known as SERRS (surface enhanced resonance Raman scattering). These principles are already known and well documented, and have been used before in the detection and analysis of various target materials.
Briefly, a Raman spectrum arises because light incident on an analyte is scattered due to excitation of electrons in the analyte. "Raman" scattering occurs when an excited electron returns to an energy level other than that from which it came--this results in a change in wavelength of the scattered light and gives rise to a series of spectral lines at both higher and lower frequencies than that of the incident light. The scattered light can be detected orthogonally to the incident beam.
Normal Raman lines are relatively weak and Raman spectroscopy is therefore too insensitive, relative to other available detection methods, to be of use in chemical analysis. Raman spectroscopy is also unsuccessful for fluorescent materials, for which the broad fluorescence emission bands (also detected orthogonally to the incident light) tend to swamp the weaker Raman emissions.
However, a modified form of Raman spectroscopy, based on "surface-enhanced" Raman scattering (SERS), has proved to be more sensitive and hence of more general use. The analyte whose spectrum is being recorded is closely associated with a roughened metal surface. This leads to a large increase in detection sensitivity, the effect being more marked the closer the analyte sits to the "active" surface (the optimum position is in the first molecular layer around the surface, ie, within about 20 nm of the surface).
The theory of this surface enhancement is not yet fully understood, but it is thought that the higher valence electrons of the analyte associate with pools of electrons (known as "plasmons") in pits on the metal surface. When incident light excites the analyte electrons, the effect is transferred to the plasmons, which are much larger than the electron cloud surrounding the analyte, and this acts to enhance the output signal, often by a factor of more than 10.sup.6. Fluorescence is also quenched, giving cleaner Raman spectra and allowing fluorescent dyes to be used as detectable analytes. Generally, the signal enhancement means that a much larger range of analytes may be usefully detected than using normal Raman spectroscopy. Furthermore, the enhancement means that a less powerful light source is required to excite the analyte molecules.
A further increase in sensitivity can be obtained by operating at the resonance frequency of the analyte (in this case usually a dye attached to the target of interest). Use of a coherent light source, tuned to the absorbance maximum of the dye, gives rise to a 10.sup.3 -10.sup.5 -fold increase in sensitivity. This is termed "resonance Raman scattering" spectroscopy.
When the surface enhancement effect and the resonance effect are combined, to give "surface enhanced resonance Raman scattering" or SERRS, the resultant increase in sensitivity and robustness is more than additive. Moreover, the sensitivity does not seem to depend so critically on the angle of orientation of the analyte to the surface, as is the case with SERS alone. A SERRS signal can be more easily discriminated from contamination and background and tends to be less variable with local conditions (eg, ionic strength or pH when an analysis is carried out in solution). SERRS is thus a surprisingly sensitive detection technique; in many instances it appears to be at least as good as, if not better than, fluorescence spectroscopy (see eg, C Rodger et al, J. Chem. Soc. Dalton Trans. (1996), pp791-799).
SERRS can also be used selectively to detect several analytes without the need for prior separation as would be necessary for fluorescence spectroscopy (see C H Munro et al in Analyst, April 1995, 120, pp993-1003).
Prior Art Relating to SERS and SERRS
SERS and SERRS have been used in the past for detecting a variety of species. Examples of relevant prior art documents include:
Appl. Spectroscopy (1993), 47, pp80-84 (J C Rubim et al)--preparation of SERS-active brass surfaces and the SERS detection of benzotriazole.
J. Raman Spectroscopy (1994), 25, pp899-901 (H Wilson et al)--SERS detection of benzotriazole deposited onto a silver colloid surface.
J. Phys. Chem. (1995), 99, pp879-885 (C H Munro et al)--use of SERRS to detect an azo dye, Solvent Yellow 14, and an explanation of the mechanisms involved.
Analyst, April 1995, 120, pp993-1003 (C H Munro et al)--SERRS detection of acidic monoazo dyes.
J. Raman Spectroscopy (1991), 22, pp771-775 (J Clarkson et al)--the effects of solvent on SERS detection of organic species on silver colloid surfaces.
U.S. Pat. No. 4,674,878 (Vo-Dinh)--ways of preparing SERS substrates, and example spectra for various organic compounds (though not nucleic acids). Detection sensitivities at nanogram and sub-nanogram levels are reported.
U.S. Pat. No. 5,400,136 (Vo-Dinh)--special coatings for SERS-active surfaces. In the examples, relatively high powered lasers are used as the light source, suggesting a fairly low level of sensitivity. Again, there is no reference to nucleic acids as target analytes.
Anal. Chem. (1990), 62, p2437-2441 (J M Bello et al)--the use of fibre optic sensors in obtaining SERS spectra. Detection limits of no lower than .about.10.sup.-7 M are quoted for various organic compounds.
Appl. Spectroscopy (1995), 49, No. 6, pp780-784 (K Kneipp et al)--detection of relatively low concentration (.about.10.sup.-16 M) of the dye rhodamine 6G, using SERRS. It should be borne in mind that this dye is likely to interact differently with a SERRS-active surface than would a Raman-labelled nucleic acid.
Mention has also been made of using SERS and SERRS to detect DNA and RNA. However, the concentrations detected have been relatively high. This suggests that prior art methods have not been sensitive enough to detect unamplified samples.
The following documents are relevant to the use of SER(R)S to detect nucleic acids:
J. Raman Spectroscopy (1991), 22, pp729-742 (T M Cotton et al)--this provides an overview of the applications of SERS and SERRS spectroscopy in biological systems. The detection of DNA is referred to, and potential problems are discussed. There is no indication of the detection sensitivities achievable in DNA analyses.
U.S. Pat. No. 5,306,403 (Vo-Dinh)--this proposes the detection of DNA by labelling with a dye and adsorbing the resulting complex onto a SERS-active surface. However, there is no enabling disclosure of a technique with sufficient sensitivity to be used without prior DNA amplification. Most of the examples relate to detection of isolated dyes, rather than of a dye-DNA complex (which, as explained below, would behave very differently under SERS conditions)--in these examples, the minimum dye concentration in the solutions investigated is 0.05 mg/ml, which probably equates to the detection of between .about.10.sup.7 and 10.sup.11 molecules. Only one example is given of the detection of a (very short) oligonucleotide labelled with aminoacridine; no concentration data is given in this example at all.
Anal. Chem. (1994), 66, pp3379-3383 (Vo-Dinh et al)--this paper reports the detection of DNA using SERS, but only at relatively high concentrations (10.sup.19 M or greater; whilst it is impossible to make exact calculations, it is unlikely that fewer than .about.10.sup.5 molecules of target were detected in the examples given). These detection levels, and the reference to the use of PCR in the paper's conclusion, indicate that the technique disclosed would still be unsuitable for detecting unamplified DNA samples.
U.S. Pat. No. 5,266,498, U.S. Pat. No. 5,376,556 and U.S. Pat. No. 5,445,972 (Tarcha et al)--these describe the detection, using SERS, of an analyte by monitoring an analyte-mediated ligand binding event. A "capture reagent" is prepared by attaching a SERS-labelled binding member, specific to the target analyte, to a SERS-active surface. Binding of the specific binding member to the analyte, in a test sample, causes a detectable change in the SERS spectrum for the capture reagent. Nucleotide sequences are briefly mentioned as possible analytes, but the documents give no example of this and no explanation as to how appropriate sensitivities might be achieved, particularly for unamplified nucleotide samples.
J. Molecular Structure (1986), 145, pp173-179 (K Kneipp et al)--SERS detection of DNA on silver sols. The DNA concentration in the experiments is .about..mu.g ml.sup.-1 ; the possibility of detecting nanogram quantities of DNA is also mentioned.
Studia Biophysica (1989), 130, pp45-50 (J Flemming et al)--again, SERS detection of DNA on silver colloid surfaces, at concentrations .about..mu.g ml.sup.-1.
Anal. Chem. (1990), 62, pp1958-1963 (F Ni et al)--investigates the possibility of combining SERS spectroscopy with flow injection analysis, to detect RNA bases at relatively high (.about.10.sup.-4 M) concentrations.
Anal. Chem. (1991), 63, pp437-442 (R Sheng et al)--use of reversed-phase high performance liquid chromatography in combination with SERS, to detect nanomolar quantities of nucleic acid bases. Sensitivity limitations are discussed, as are possible ways of overcoming them.
J. Molecular Structure (1991), 244, pp183-192 (K Kneipp et al)--SERS detection of various nucleic acids, including DNA and RNA, at concentrations no lower than .about.10 .mu.g ml.sup.-1.
Appl. Spectroscopy (1994), 48, pp951-955 (K Kneipp et al)--near-infrared SERS detection of the DNA base adenine adsorbed onto silver or gold colloidal particles. The lowest base concentration detected is 10.sup.-7 M.
Thus, earlier experiments have in common the fact that they use relatively large quantities of nucleic acid analyte. None has yet demonstrated sensitivities high enough to allow the detection of unamplified nucleic acid samples (ie, the detection of perhaps 1-100 molecules in a sample).
That SERS and SERRS have never been proposed for use in the detection of unamplified nucleic acids is due at least in part to the obvious difficulties in achieving the appropriate sensitivities. These difficulties are partly due to problems specific to nucleic acids, problems which are therefore not addressed in the more general SER(R)S literature.
The skilled person seeking to detect nucleic acids or nucleic acid units would thus consider SER(R)S spectroscopy to lack the necessary sensitivity or robustness, certainly without target amplification. The need remains for an alternative detection method, suitable for use with very low concentrations of target, and this is what the present invention provides.